C-Terminal Binding Proteins: Central Players in Development and Disease

BioMol Concepts 2014; 5(6): 489–511 Review Trisha R. Stankiewicz, Josie J. Gray, Aimee N. Winter and Daniel A. Linseman* C-terminal binding proteins: central players in development and disease Abstract: C-terminal binding proteins (CtBPs) were initially Introduction identified as binding partners for the E1A-transforming proteins. Although the invertebrate genome encodes one C-terminal binding protein (CtBPs) were originally identi- CtBP protein, two CtBPs (CtBP1 and CtBP2) are encoded fied as binding partners for the adenovirus E1A protein, by the vertebrate genome and perform both unique and suggesting an important function for CtBPs in regulat- duplicative functions. CtBP1 and CtBP2 are closely related ing cell survival (1, 2). CtBPs are evolutionarily conserved and act as transcriptional corepressors when activated by proteins and are present in a diverse number of species nicotinamide adenine dinucleotide binding to their dehy- ranging from terrestrial plants to Caenorhabditis elegans drogenase domains. CtBPs exert transcriptional repres- to humans (3). Whereas the vertebrate genome encodes sion primarily via recruitment of a corepressor complex two highly homologous CtBP proteins, CtBP1 and CtBP2, to DNA that consists of histone deacetylases (HDACs) the invertebrate genome encodes one CtBP transcript. The and histone methyltransferases, although CtBPs can also major splice forms of CtBP1 and CtBP2 function primar- repress transcription through HDAC-independent mecha- ily as transcriptional corepressors. CtBPs associate with nisms. More recent studies have demonstrated a critical various DNA-binding repressors such as basic Krüppel- function for CtBPs in the transcriptional repression of like factor 8 (BKLF8) to recruit chromatin-modifying pro-apoptotic genes such as Bax, Puma, Bik, and Noxa. enzymes to the promoter regions of DNA (4, 5). However, Nonetheless, although recent efforts have characterized some minor CtBP splice variants also mediate various the essential involvement of CtBPs in promoting cellular cytosolic functions (3). survival, the dysregulation of CtBPs in both neurodegen- Genetic knockout studies have demonstrated that erative disease and cancers remains to be fully elucidated. CtBP1 and CtBP2 perform both unique and duplicative functions during development and genetic deletion of Keywords: anoikis; apoptosis; cancer; C-terminal binding CtBPs results in severe developmental defects and embry- proteins; development; neurodegenerative disease. onic lethality (6). Consistent with their role in transcriptional corepression, Grooteclaes and colleagues (4, 7) DOI 10.1515/bmc-2014-0027 identified CtBPs as critical repressors of apoptotic and Received August 11, 2014; accepted October 7, 2014 anoikis gene programs. Subsequent studies have demonstrated that CtBPs bind the viral oncoproteins EBNA3A and EBNA3C, further demonstrating that CtBPs have significant roles in regulation of cellular proliferation and survival (8, 9). Given the critical functions of CtBPs in promoting cell survival, dysregulation of CtBPs has emerged *Corresponding author: Daniel A. Linseman, Research Service, as a potential causative factor underlying neurodegenera- Veterans Affairs Medical Center, Research 151, 1055 Clermont tive diseases as well as cancer. Street, Denver, CO 80220, USA, e-mail: [email protected]; In this review, we summarize current information on Department of Biological Sciences and Eleanor Roosevelt Institute, the structure, function, and regulation of CtBPs. Next, University of Denver, Denver, CO 80208, USA; and Division of Clinical Pharmacology and Toxicology, Department of Medicine and we discuss the involvement of CtBPs in development, Neuroscience Program, University of Colorado Denver, Aurora, CO apoptosis, and anoikis. Finally, we highlight recent data 80045, USA supporting dysregulation of CtBPs in neurodegenera- Trisha R. Stankiewicz and Josie J. Gray: Research Service, Veterans tive diseases and cancer. Given the extensive evidence Affairs Medical Center, Denver, CO 80220, USA; and Department of demonstrating a pro-survival function for CtBPs in both Biological Sciences and Eleanor Roosevelt Institute, University of Denver, Denver, CO 80208, USA non-neuronal and neuronal cell populations, CtBPs are Aimee N. Winter: Department of Biological Sciences and Eleanor emerging as novel molecular targets for the treatment of Roosevelt Institute, University of Denver, Denver, CO 80208, USA diseases characterized by either enhanced apoptosis (i.e., 490 T.R. Stankiewicz et al.: CtBPs in development and disease neurodegeneration) or increased resistance to apoptotic CtBP to execute its corepressor functions (10). CtBPs can cell death (i.e., cancer). also bind to proteins directly via the PXDLS domain to repress their function (15). In addition to the PXDLS-binding pocket, all CtBP family members also contain a surface groove that is Structure and function of CtBPs capable of binding proteins with an RRT-binding motif (16). Like the PXDLS hydrophobic cleft, the RRT-binding Domain structure of CtBPs pocket of CtBPs is predominantly used to bind and recruit members of the corepressor complex, and many CtBP- CtBP1 and CtBP2 are highly conserved and share a sig- interacting proteins (CtIPs) that bind at this site also nificant degree of sequence and structural homology contain PXDLS-binding motifs (10, 16). Thus, each CtBP (Figure 1). Of these two proteins, CtBP1 has been the most monomer contains two binding sites that can be occupied extensively characterized. Although CtBP1 and CtBP2 simultaneously by distinct members of the corepressor possess high sequence homology and structural simi- complex. Together, these two binding sites account for larities, they have been shown to exert both unique and a majority of CtBP interactions with proteins of the core- duplicative effects (6). Despite these diverse functions, pressor complex and repressor proteins that recruit CtBPs however, all CtBP family members share several key char- to specific gene promoters. acteristics that are essential for protein function. Intriguingly, it is now well established that CtBP A hydrophobic cleft known as the PXDLS-binding site proteins share significant sequence homology with 2D is one of the best characterized features of the CtBP family hydroxyacid dehydrogenases, particularly within the RTT- and several CtBP-interacting partners containing PXDLS- binding cleft (Figure 1) (17). Moreover, the dehydrogenase binding motifs have been identified. In many cases, muta- domain contains a dinucleotide-binding site that is capable tions in either the PXDLS-binding pocket in CtBPs or in of binding both NAD+ and nicotinamide adenine dinucleo- the binding motif of interacting proteins is sufficient to tide (NADH) (17). Although CtBPs are functional dehydroge- abrogate their interaction (2, 10–14). Moreover, this hydro- nases, the significance of this observation is not yet known, phobic cleft is essential to CtBP function because it plays and the physiological substrate for CtBP deacetylase activ- an extensive role in recruiting other members of the CtBP ity has not been identified (18). However, it has been estab- corepressor complex in both a PXDLS-dependent and a lished that the ability of CtBPs to bind NADH is required for PXDLS-independent manner (10). Specifically, the PXDLS- these proteins to form dimers with one another and exert binding site is essential for recruiting the core machinery transcriptional repression (10, 18–20). Nonetheless, CtBP of the corepressor complex, including histone deacety- mutants that are deficient in their ability to form dimers do lases (HDACs), histone methyltransferases (HMTases), display the ability to mediate other CtBP functions, which and transcriptional repressors, which are required for are discussed in further detail below (10). Figure 1 Structures of CtBP1 and CtBP2. The PXDLS-binding cleft, RRT-binding cleft, and dehydrogenase domain of CtBP1 and CtBP2 compose the majority of the CtBP protein structure. Note the longer N-terminus of CtBP2, which contains an NLS that is absent from CtBP1. Also note that CtBP1 contains a PDZ domain at the C-terminus that is absent from CtBP2. T.R. Stankiewicz et al.: CtBPs in development and disease 491 Collectively, the PXDLS-binding cleft, RRT-binding cleft, and dehydrogenase domain of CtBP1 and CtBP2 compose the majority of the CtBP protein structure and are highly homologous for both CtBP family members and their various isoforms (Figure 1). However, there are also slight differences in protein sequence that contribute to the differing functions of CtBP1 and CtBP2. Most notably, CtBP2 contains a nuclear localization signal (NLS) at its N-terminus that is absent in CtBP1 (21). This region of CtBP2 contains several lysine resi- dues that are acetylated, contributing to the nuclear reten- tion of this protein (22). The NLS is absent in some isoforms of CtBP2, resulting in the accumulation of a small cytoplasmic pool of this protein; however, the cytoplasmic function of CtBP2 remains unclear. In contrast, CtBP1 relies on binding Figure 2 Transcriptional regulation by CtBPs. to transcriptional repressors such as zinc finger E-box pro- As CtBPs do not bind to DNA directly, they recruit various chromatin- teins (ZEB1/2), SUMOylation, and other interactions such as modifying enzymes to exert transcriptional repression. CtBPs can heterodimerization with CtBP2 for nuclear import and reten- recruit a corepressor complex consisting of HDAC1/2, Ubc9, and tion

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